Isotopes are atoms which have different masses due to changes in the number of neutrons in their nuclei. One of the most widely used stable isotopes in the pharmaceutical industry is deuterium (D; 2H), an isotope of hydrogen with a nucleus comprising one neutron and one proton. A stable isotope is one which does not undergo radioactive decay. Deuterium was discovered as a natural occurring isotope in H2O, which contains approximately 0.015% deuterium in the form of “heavy water” (D2O). Its use as a moderator in nuclear reactors, initially provided impetus for its large-scale manufacture. However, all D2O production processes require large amounts of energy, so that its cost has remained high. Some of the physical properties of D2O include greater density and viscosity than H2O and a higher melting and boiling point; whereas differences in various other physical properties are less marked. The differences between a deuterated and parent compound, also called the protio molecule, is exploited in drug discovery programs through isotopic labeling techniques to better understand mechanism of action, as well as to identify and quantify metabolites in an effort to understand metabolism-mediated toxicities.
Stable isotope-labeled compounds have been employed in several areas of biomedical research. The combination of stable isotope-labeling techniques with mass spectrometry (MS), which allows rapid acquisition and interpretation of data, has promoted greater use of these stable isotope-labeled compounds in a number of fields including absorption, distribution, metabolism, and excretion (ADME) studies. The use of stable isotope labeling to study various aspects of the metabolism and pharmacokinetics of drugs and other foreign compounds in animals and humans has been well-documented. See, e.g., Zhu, M., et al., Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data. Drug Metab. Dispos. 34:1722-1733 (2006).
Compounds labeled with stable isotopes, such as deuterium and 13C, have been used effectively in the past by drug metabolism scientists and toxicologists to gain a better understanding of a drug's disposition and its potential role in target organ toxicities. Other quantitative applications of stable isotope-labeled compounds include studies conducted to distinguish in vivo and in vitro disposition of enantiomers where only one of the enantiomers was selectively labeled with stable isotopes. See, e.g., Eichelbaum, M., et al., Application of stable labeled drugs in clinical pharmacokinetic investigations. Clin. Pharmacokinet. 7:490-507 (1982). In another study, a stable isotope-labeled glucuronide conjugate of acetaminophen was used to explain the results of in vitro kinetic data. See, e.g., Mutlib, A. E., et al., Kinetics of acetaminophen glucuronidation by UDP Glucuronosyltransferases 1A1, 1A6, 1A9 and 2B15. Potential implications in acetaminophen-induced hepatotoxicity. Chem. Res. Toxicol. 19:701-709 (2006). Despite the greater availability of stable isotope-labeled compounds, drug metabolism scientists have yet to take full advantage of the potential use of these analogues for mechanistic metabolism and toxicity studies. These stable isotope-labeled compounds can be used to gain a better understanding of a drug's disposition and in toxicity studies. Identification of metabolite structures is very important, especially if one is trying to understand metabolism-mediated toxicities.
Toxicogenomics is a rapidly evolving field and is expected to play a very significant role in drug discovery and development in future. However, while significant progress has been made in toxicogenomics techniques, the interpretation of large sets of data produced from these studies can be a challenge. One approach that could be used to simplify interpretation of the data, especially from studies designed to link gene changes with the formation of reactive metabolites thought to be responsible for toxicities, is through the use of stable isotope-labeled compounds. The employment of analytical techniques, especially mass spectrometry and NMR used in conjunction with stable isotope-labeled compounds to establish and understand the mechanistic link between reactive metabolite formation, genomic and proteomic changes, and the onset of toxicity, appears very logical. This interdisciplinary approach may provide potential genomic and/or proteomic biomarkers of target organ toxicities, within the near future.
The greater availability of stable isotope-labeled analogues, especially synthesized to be used as internal standards for quantitative studies, has made it possible to use these compounds to conduct mechanistic metabolism studies. Often, a 1:1 mixture of labeled and unlabeled compound is used to create recognizable mass spectral ion patterns showing the presence of drug-related materials in complex biological mixtures. Technology has advanced to a point where a combination of mass spectrometry (MS) and stable isotope-labeled compounds can be used to provide a wealth of information on the metabolic disposition and identities of metabolites in the absence of radiolabeled compounds or authentic metabolite standards.
In addition, various other analytical techniques, such as nuclear magnetic resonance (NMR) spectroscopy, may be more widely used in conjunction with stable isotope-labeled compounds and mass spectrometry to better understand metabolic disposition and in elucidating structures of metabolites. Recent advancements in NMR technology that have allowed significant gains in sensitivity will make this methodology even more amenable in the determination of structures of metabolites of compounds labeled with stable isotopes. Strategic placement of stable isotope label(s) in a compound can also allow a better understand some of the gene changes attributed to reactive metabolite formation and/or to a particular metabolic pathway. Target organ toxicities can be modulated by selective introduction of stable isotopes, such as deuterium, in a molecule. Studying and comparing gene changes produced by labeled and nonlabeled compounds can provide an idea of critical genes that may be involved in the onset of toxicities. This is an area of intensive research, in the attempt to obtain “signature” genes that could be used as biomarkers for specific target organ toxicities.
Furthermore, one can use stable isotope-labeled compounds to delineate potential metabolism-mediated toxicities. If one suspects that a particular metabolic pathway or a metabolite is involved in causing toxicity by in situ generation of toxic metabolites as latently reactive species or “shunt-products” in vivo, stable isotope labels can be placed in such a manner by strategically substituting acidic protons by deuterium as to modulate the formation of the specific metabolite, hence potentially mitigating the toxicity. Obviously, one can conduct in vitro studies with labeled and nonlabeled compounds to understand the effect of labeling (e.g., the kinetic/deuterium isotope effect, which will be discussed infra) on the formation of a metabolite before an extensive toxicity study is conducted. Having stable isotope “labels” on reactive intermediates can greatly assist the identification of sites on proteins modified through covalent binding. Studies can be designed to investigate if particular proteins are targeted by reactive intermediates using stable isotope-labeled compounds. Studies encompassing the simultaneous use of radio- and stable isotope-labeled compounds to study proteomic and genomic changes as a consequence of reactive metabolite-mediated toxicity should potentially lead to a better understanding of some target organ toxicities and perhaps may lead to the identification of potential genomic or proteomic biomarkers.
Living systems exposed to D2O experience at least two sets of effects. One is a “solvent isotope effect”, due to the properties of D2O itself, and especially its effects on the structure of water and macromolecules. The second is the “kinetic isotope effect” (KIE), resulting from the ability of D2O to replace H with D in biological molecules. In general, the C-D bond is about 10-times as strong as the C—H bond and is more resistant to chemical or enzymatic cleavage. Thus, compounds with C-D bonds tend to remain stable in H2O indefinitely, and such compounds have been very widely used for isotopic studies. O-D, N-D and S-D bonds are also stronger than the corresponding protonated forms, but the D in such bonds quickly exchanges with H in H2O especially when the deuterated position in the molecule is chemically labile for deuterium scrambling or rearrangements. See, e.g., Thomas, A. E 1971. Deuterium labeling in organic chemistry. Appleton-Century Crofts: New York. Deuterium isotope effects are usually considered in terms of D linkages to C atoms. Deuteration of O, N and S in biological molecules must occur rapidly when the cells are exposed to D2O but the reversibility of these processes by exchange with H+ makes it very difficult to assess the biological effects of such deuteration. The ratio of the rates of cleavage of a C-protonated and D-deuterated compound, expresses the “primary” deuterium isotope effect, usually called simply the kinetic isotope effect (KIE). See, e.g., Foster, A. B., Deuterium isotope effects in the metabolism drugs and xenobiotics: implications for drug design. Adv. Drug Res. 14:1-40 (1985). Ten-fold differences in reaction rates are common.
“Secondary” deuterium isotope effects occur when attachment of deuterium to another atom affects the rate of C—H cleavage; such effects are usually small. The existence of a DIE in comparing protonated and deuterated compounds has been widely used to show whether metabolic reactions involve cleavage of 13C bonds. For example, this technique was used by Deraaiyagala, et al., (β-Secondary and solvent deuterium kinetic isotope effects and the mechanisms of base- and acid-catalyzed hydrolysis of penicillanic acid. J. Org. Chem. 60:1619-1625 (1995)) and by Paterson, et al., (An antibody binding site on cytochrome C defined by hydrogen exchange and two dimensional MNMR. Science 249:755-759 (1990) to study the mechanisms of antigen-antibody reactions. The use of deuterium labels in many spectroscopic studies (for a review see, e.g., Kushner, D. J., et al., Biotechnological potential of heavy water and deuterated compounds. Proceedings of Biotechnology Risk Assessment Symposium. Ottawa, Canada. Jun. 13-15, 1996. Edited by Levin. C. and J. S. Angle. University of Maryland Biotechnology Institute Publication 1003. pp. 75-89 (1997)) illustrate the important contributions this isotope has made to current biological and pharmacological research. These aforementioned kinetic isotopic effects will be discussed more fully, below.
I. Deuterium Kinetic Isotope Effect
The deuterium kinetic isotope effect (KIE) is a dependence of the rate of a chemical reaction on the isotopic identity of an atom in a reactant and is observed in a change of the rate of reaction that occur when deuterium is substituted for hydrogen. By way of example, the KIE involving hydrogen and deuterium may be represented by the equation:
      KIE    =                  k        H                    k        D              ;wherein kH and kD are reaction rate constants for hydrogen and deuterium, respectively.
The deuterium-mediated isotope effects result from the greater energy required to break a covalent bond to deuterium versus a covalent bond to hydrogen, and occur because of the significant mass difference between hydrogen and deuterium. The C-D bond is up to 10-times stronger than the C—H bond, making it more resistant to chemical or enzymatic cleavage. An isotopic substitution will greatly modify the reaction rate when the isotopic replacement is in a chemical bond that is broken or formed in the rate limiting step. In such a case, the change is termed a primary isotope effect. When the substitution is not involved in the bond that is breaking or forming, a smaller rate change, termed a secondary isotope effect is observed. Thus, the magnitude of the kinetic isotope effect can be used to elucidate the specific reaction mechanism. However, if other steps are partially rate-determining, the effect of isotopic substitution will be masked.
Isotopic rate changes are most pronounced when the relative mass change is greatest since the effect is related to vibrational frequencies of the affected bonds. For example, changing a hydrogen atom to deuterium represents a 100% increase in mass; whereas in replacing carbon-12 (12C) with carbon-13 (13C), the mass increases by only 8%. Therefore, the rate of a reaction involving a C—H bond is typically 6- to 10-times faster than the corresponding C-D bond. Moreover, the C-D bond is up to 10-times stronger than the C—H bond, making it more resistant to chemical or enzymatic cleavage. In contrast, a 12C reaction is only approximately 1.04-times faster than the corresponding 13C reaction (even though, in both cases, the isotope is one atomic mass unit heavier).
Isotopic substitution can modify the rate of reaction in a variety of ways. In many cases, the rate difference can be rationalized by noting that the mass of an atom affects the vibrational frequency of the chemical bond that it forms, even if the electron configuration is nearly identical. Heavier atoms will (in a classical mechanical analysis) lead to lower vibration frequencies or, in a quantum mechanical analysis, will have lower zero-point energy. The zero-point energy is the lowest possible energy that a quantum mechanical physical system can have, and is the energy of the ground state. With a lower zero-point energy, more energy must be supplied to break the bond, resulting in a higher activation energy for bond cleavage, which in turn lowers the measured rate. The rate of a chemical reaction may be calculated using, e.g., the Arrhenius equation.
The Arrhenius equation is a simple, but accurate, formula for the temperature dependence of the reaction rate constant, and therefore, the overall rate of a chemical reaction. In short, the Arrhenius equation gives the dependence of the rate constant “k” of a chemical reaction at the temperature “T” (in absolute temperature, such as degrees Kelvin or Rankine) and activation energy “Ea”, as shown below:k=Ae−a/RT;wherein “A” is the pre-exponential factor or simply and “R” is the gas constant. The units of the pre-exponential factor are identical to those of the rate constant and will vary depending on the order of the reaction. If the reaction is first order it has the units s−1, and for that reason it is often called the frequency factor or attempt frequency of the reaction. Most simply, k is the number of collisions which result in a reaction per second, A is the total number of collisions (leading to a reaction or not) per second and e−Ea/RT is the probability that any given collision will result in a reaction. When the activation energy is given in molecular units instead of molar units (e.g., joules) per molecule instead of joules per mole, the Boltzmann constant is used instead of the gas constant. It can be seen that either increasing the temperature or decreasing the activation energy (for example through the use of catalyst) will result in an increase in rate of reaction.
Given the small temperature range in which kinetic studies are carried, it is reasonable to approximate the activation energy as being independent of the temperature. Similarly, under a wide range of practical conditions, the weak temperature dependence of the pre-exponential factor is negligible compared to the temperature dependence of the exp(−Ea/RT) factor; except in the case of “barrierless” diffusion-limited reactions, in which case the pre-exponential factor is dominant and is directly observable.
The Arrhenius equation states that the fraction of molecules that have enough energy to overcome an energy barrier, that is, those with energy at least equal to the activation energy, depends exponentially on the ratio of the activation energy to thermal energy (RT), the average amount of thermal energy that molecules possess at a certain temperature. The transition state in a reaction is a short lived state (on the order of 10−14 sec) along the reaction pathway during which the original bonds have stretched to their limit. By definition, the activation energy (Ea) for a reaction is the energy required to reach the transition state of that reaction. Reactions that involve multiple steps will necessarily have a number of transition states, and in these instances, the activation energy for the reaction is equal to the energy difference between the reactants and the most unstable transition state. Once the transition state is reached, the molecules can either revert, thus reforming the original reactants, or new bonds form giving rise to the products. This dichotomy is possible because both pathways, forward and reverse, result in the release of energy. A catalyst facilitates a reaction process by lowering the activation energy leading to a transition state. Enzymes are examples of biological catalysts that reduce the energy necessary to achieve a particular transition state.
A carbon-hydrogen bond is by nature a covalent chemical bond. Such a bond forms when two atoms of similar electronegativity share some of their valence electrons, thereby creating a force that holds the atoms together. This force or bond strength can be quantified and is expressed in units of energy, and as such, covalent bonds between various atoms can be classified according to how much energy must be applied to the bond in order to break the bond or separate the two atoms. Bond strength is directly proportional to the absolute value of the ground-state vibrational energy of the bond. This vibrational energy, which is also known as the zero-point vibrational energy, depends on the mass of the atoms that form the bond. The absolute value of the zero-point vibrational energy increases as the mass of one or both of the atoms making the bond increases. Since deuterium (D) has twice the mass of hydrogen (H), it follows that a C-D bond is stronger than the corresponding C—H bond. Compounds with C-D bonds are frequently indefinitely stable in H2O, and have been widely used for isotopic studies. If a C—H bond is broken during a rate-determining step in a chemical reaction (i.e., the step with the highest transition state energy), then substituting a deuterium for that hydrogen will cause a decrease in the reaction rate and the process will slow down. As previously discussed, this is known as the deuterium kinetic isotope effect (KIE). The magnitude of the KIE can be expressed as the ratio between the rates of a given reaction in which a C—H bond is broken, and the same reaction where deuterium is substituted for hydrogen. The KIE can range from about 1 (i.e., no isotope effect) to very large numbers (i.e., ≧50), meaning that the reaction can be fifty, or more, times slower when deuterium is substituted for hydrogen. High KIE values may be due in part to a phenomenon known as tunneling, which is a consequence of the uncertainty principle. Tunneling is ascribed to the small mass of a hydrogen atom, and occurs because transition states involving a proton can sometimes form in the absence of the required activation energy. Because deuterium has more mass than hydrogen, it statistically has a much lower probability of undergoing this phenomenon. Substitution of tritium for hydrogen results in yet a stronger bond than deuterium and gives numerically larger isotope effects.
If the cleavage of a C—H bond is implicated in the rate-determining step of a metabolic pathway, an overall decrease in metabolism will be observed when hydrogen is substituted with deuterium. Therefore, the reduction in metabolism attributable to deuterium substitution extends the desired effects of a drug while retarding its undesirable effects. One of the challenges of incorporating deuterium into a drug is the possibility of deuterium/hydrogen exchange within the physiological environment, which tends to eviscerate the effect of the compound. Further, when deuterium retards metabolism at one site, a phenomenon called “metabolic switching” or “metabolic shunting” can occur. The suppression of one metabolic pathway promotes metabolism at another site which quantitatively changes the paths of metabolism of the drug.
For a deuterated clinical candidate to be successful, it must address the problems of biochemical deuterium exchange and metabolic switching. The ideal starting point in developing a deuterated drug, also referred to as an isotopolog, is to selectively deuterate a drug in clinical development which has a known metabolic profile. Deuterated drugs of interest are those whose pharmacological or metabolic profiles differ from their protonated versions.
It should also be noted, however, that reactions are also known where the deuterated species reacts faster than the non-deuterated analog, and these cases are said to exhibit inverse kinetic isotope effects (IKIE). IKIEs are often observed in the reductive elimination of alkyl metal hydrides, e.g., Me2NCH2CH2NMe2)PtMe(H). In such cases, the C-D bond in the transition state, an agostic species, is highly stabilized relative to the C—H bond.
II. Effects of D2O on Proteins, Cells and Tissues
As a solvent, D2O increases stability of proteins and other molecules, likely through increasing the formation of hydrophobic bonds. The effect of D20 on hydrophobic bond formation was thought to cause stabilization of heliozoan microtubule formation, and it has been used as an active polymerizer of tubulin in a number of systems. See, e.g., Sollott, S. J., et al., Taxol inhibits neointinal smooth muscle cell accumulation after angioplasty in the rat. J. Clin. Invest. 95 1869-1876 (1995).
The anti-mitotic action of D2O has stimulated its use as an antitumor agent. Effective D2O concentrations were usually too toxic to animals for rational chemotherapy. Combining D2O treatment with cytotoxic drugs such as methotrexate caused more reduction of tumor growth than either agent alone, although definitive cures did not result (Laissue, et al. 1982). A more recent study (Bauer, et al. 1995) showed that D2O was much more effective in killing malignant melanoma and carcinoma cells (colon carcinoma, glioblastoma, and small lung cell cancer cells) than PHA-stimulated lymphocytes and normal glial cells. For example, 90% D2O was shown to kill 70% of the former, but only 5% of the latter group. A differential effect on cell growth also occurred and 9 days of treatment with 90% D2O reduced the viable fraction of malignant cells to about 0.1%. Again. the effective D2O concentrations were too high for use in human therapy.
D2O inhibits mitosis in many plant and animal cells. This effect seems due partly to its effect on tubulin polymerization and also, or especially, on its action on microtubule organizing centers and other structures governing formation of the mitotic spindle (Lamprecht, et al. 1991). Other effects of D2O on cell structure have also been noted. In addition to affecting the formation of different blood cells, including platelets, D2O also affects platelets in vitro, inhibiting their spreading, retraction, and aggregation by ADP and collagen (Adains and Adanls 1988); as well as stimulating their adrenaline-induced aggregation (Reuter, et al. 1985). While these effects on platelet movement were discussed in terms of membrane receptors and energy metabolism, the effects of D2O on microfilament systems, which may be responsible for changes in shape of human neutrophil granulocytes (Zimmennann, et al. 1988), might also be involved in the effects on platelets.
Vasilescu and Karoila (1986) found that D2O inhibited bioelectrogenesis and contractility in nerve and muscle preparations and uncoupled electrical and mechanical functions in the isolated frog heart. It also lowered the ATP/ADP ratio in these tissues and also played an antagonistic role to anesthetics in sciatic nerve trunk. It was also shown that D2O only slightly inhibited sodium transport activity in human leucocytes. D2O competition may have important effects on calcium channel activity. It has been suggested that the anti-hypertensive effects of D2O may be related to its ability to reduce L-type calcium channel conductance in myocytes and calcium uptake in rat aortic rings treated with phenylephrine and KCl. D2O has a number of other effects on membrane function; including membrane depolarization and activation of calcium channels in algae, inhibition of Na+—K+ ATPase in membranes and interference with H+ exchange in hepatic cells.
III. Effects of D2O on the Metabolism of Drugs
As previously stated, the C-D bond is more stable than the C—H bond, and once incorporated into organic compounds, deuterium is not readily exchangeable in H2O. Deuterated organic compounds can be detected with great sensitivity by mass spectrometry and other methods. Because of these considerations, and the generally very low toxicity of deuterated compounds (especially compared with radioactive ones), such deuterated drugs are very widely used in studies of metabolism and movement of drugs and other substances in humans and other animals.
Deuteration of pharmaceuticals to improve pharmacokinetics (PK), pharmacodynamics (PD), and toxicity profiles, has been demonstrated previously with some classes of drugs. However, this method may not be applicable to all drug classes. For example, deuterium incorporation can lead to metabolic switching. The concept of metabolic switching asserts that xenogens, when sequestered by Phase I enzymes, may bind transiently and re-bind in a variety of conformations prior to the chemical reaction (e.g., oxidation). This hypothesis is supported by the relatively vast size of binding pockets in many Phase I enzymes and the promiscuous nature of many metabolic reactions. Metabolic switching can potentially lead to different proportions of known metabolites as well as altogether new metabolites. This new metabolic profile may impart more or less toxicity. Such pitfalls are non-obvious and are not predictable a priori for any drug class.
There are, however, multiple specific examples of deuterium's effect on the metabolism of biologically active molecules. For example, the anesthetic chloroform (CHCl3) is metabolized in vivo to phosgene, a highly reactive alkylating agent. Deuteration of chloroform to deuterochloroform (CDCl3) decreases its metabolic rate, thereby reducing liver and lung toxicity in rats by up to 70% over chloroform. Conversely, 1,2-dibromoethane (ClCH2CH2Cl) is itself a DNA alkylating species, and the tetra-deuterated analog (ClCD2CD2Cl) is found to be metabolized markedly more slowly than the protio version. However, the deuterated species actually causes more DNA damage than its protio counterpart because reduced metabolism prolongs the existence of the reactive species in the body.
Deuteration can also reduce a drug's pharmacological activity. An example is the anti-anxiety drug Valium® (diazepam), which requires 3-hydroxylation to oxepam for its anticonvulsive action. Diazepam, which is di-deuterated at position 3, has lower anticonvulsive action which may be due to the lower degree of 3-hydroxylation.
Deuterated analogs of various drugs including, but not limited to: the electron-affinic radiosensitizers and antitumor agents—RSU 1069 and Ro 03-8799; neurotoxill MPTP (1-inethyl-4-phenyl-1,2,3,6-tetrahydi-opyridine); nordiazepain; amines; nonsteroidal anti-inflammatory 2-arylpropionic acids; anti-malarial drugs; penicillamine; and the like have been synthesized and studied. For a more complete listing of various deuterated drugs, see, e.g., Yarnell, A., Heavy Hydrogen Drugs Turn Heads, Again. Chem. Engineer. News June 22:36-38 (2009).
Of special interest are drugs that are metabolized by the hepatic cytochrome P450 system, and the monooxygenases that act on various types of compounds. One of the first steps in all such reactions is the breaking of a C—H bond; and compounds that have C-D structures at the site of enzymatic attack are more resistant to P450-induced change. Resistance to P450-induced changes may lead to an increase in duration of pharmacological action or other desired properties. For example, tamoxifen is widely utilized in the treatment of human breast cancer, but it has also been shown to be capable of causing liver cancers in rats. This is thought to be related to a hydroxylation of part of the tamoxifen molecule, converting it to a DNA adduct. This hypothesis is supported by findings that deuterated tamoxifen, which has lower hepatotoxicity than the hydrogenated form, was also less susceptible to hydroxylation (Jarman et al. 1995).
Incorporating deuterium into novel compounds in an effort to mediate metabolism is a strategy which is finding success in traditional drug design and development. While deuterium has been extensively used as a tool to identify metabolites and metabolic pathways, it has only just recently been incorporated into several clinical candidates in Phase 1 drug development programs targeting deuterated analogs of small molecules in an effort to alter their metabolic profiles. Initial results from the clinical trials of deuterated Effexor® and Paxil® analogs, demonstrate the potential of deuterating known drugs, as both trials exhibited a reduction in the metabolism of the aforementioned deuterated compounds. See, Yarnell, A., Heavy Hydrogen Drugs Turn Heads, Again. Chem. Engineer. News June 22:36-38 (2009). More specifically, an example of the differences between an isotopolog and its protio version is shown in the clinical trial data of SD-254, an isotopolog of Effexor®. SD-254 was found to be metabolized half as fast as Effexor®, and pharmacologically-effective levels of the drug were maintained after 24 hours, substantially longer than that observed for the protio version. See, Id. This difference in the pharmacokinetics of SD-254 may allow for the administration of a lower dose while maintaining the same effects, thereby decreasing the incidences of deleterious side effects, which are generally dose-related.
It should be noted, however, that recent FDA guidance on the safety testing of metabolites will probably lead some investigators to revisit the application of stable isotope-labeled compounds in absorption, distribution, metabolism, and excretion (ADME) studies. The ability to demonstrate human-specific metabolite coverage in preclinical species as early as possible has become a challenge with the issuance of this guidance. Hence, identification of major human metabolites (considered to be greater than 10% of parent AUC values) during early drug development has become very important. The administration of a 1:1 mixture of labeled and nonlabeled analogues is one approach that will enable researchers and drug developers to rapidly identify all drug-related components in the plasma of humans during early stages of drug development. Even though major progress has been made in the field of mass spectrometry in detecting and identifying metabolites, one can still possibly miss unexpected or unusual metabolites using the existing LC/MS technology. The appearance of twin ion pairs in the mass spectra of plasma extracts can be used to scan for all possible metabolites in circulation in the absence of synthetic metabolite standards or radiolabeled compounds. Additionally, the application of LC-CRIMS (liquid chromatography-chemical reaction interface mass spectrometry) in combination with stable isotope-labeled compounds to obtain both qualitative and quantitative information on metabolites of potential therapeutic agents administered in early human studies also may be increasingly utilized.